Project Summary Protein aggregation is a central feature of aging and aging-associated degenerative diseases. Various lines of evidence suggest that aging-correlated protein aggregates, typically composed of intrinsically disordered proteins, are themselves toxic. Other data suggests that large aggregates (e.g., amyloid fibers) are neuroprotective, while protofibrils are the toxic species. Finally, some research suggests that aggregates are not themselves toxic, but that they have indirect effects that are problematic for cell health. These contrasting perspectives demand further studies to delineate the biophysical and mechanistic consequences of protein aggregation in the setting of intact cells, tissues, and organs. In preliminary work, thermal proteome profiling experiments using gel-based assays suggested that the presence of aggregates in cells catalytically destabilizes proximal proteins, enhancing their likelihood of irreversible unfolding. This observation suggests a new hypothesis for why aggregates engender diverse phenotypes, but are nonetheless universally associated with cellular degeneration. Specifically, it is proposed that selective precipitation of components of the proteome catalyzed by the presence of protein aggregates underpins their cytotoxicity, and sensitizes aging cells to stress. A detailed evaluation of this mechanism requires quantitative mapping in living cells of the proteome components sensitized to catalytic destabilization by the presence of proximal early- and late-stage protein aggregates. To enable this objective, thermal proteome profiling using quantitative, proteome-wide mass spectrometry is here employed for the first time to simultaneously determine protein stability curves for thousands of individual proteins in the context of the expression of various aggregating proteins in living cells. Experiments performed using this method will directly test the hypothesis that aggregates destabilize otherwise well-behave proteome components critical for normal cell function, and will determine which components of the proteome are most destabilized by the presence of aggregates. Additionally, chemical biology tools to regulate the proteostasis network will be deployed to test whether modulating chaperone levels can ameliorate aggregate proximity-induced protein destabilization. Mechanistic follow-up studies will confirm top hits and provide insight into why aggregate-induced proteome destabilization induces cytotoxic effects.